Hereditary cancer syndromes account for a small percentage of all cancers diagnosed each year, although they are common in some cancer forms, such as colorectal and breast cancers (1, 2). Colorectal cancer (CRC) is considered one of the most frequent malignancies worldwide, and a family history of the condition is a major risk factor (1, 3). Notably, the relative risk of developing CRC increases in individuals with a family history of CRC. Alongside screening and surveillance, another advantage of determining the molecular basis of familial CRC is its therapeutic relevance (4, 5). Meanwhile, chromosomal instability arises in a remarkable portion of CRC tumors. Microsatellite instability (MSI), which is caused by the aberrant function of the DNA mismatch repair (MMR) system, accounts for nearly 15% of all CRC patients. In fact, insertion or deletion of a repetitive unit in a short tandemly repeated sequence (STR), designated as MSI, is a robust diagnostic marker for identifying Lynch syndrome (LS) suspicious individuals (6–8).

LS is an autosomal dominant hereditary cancer syndrome caused by germline defects in the DNA mismatch repair genes, including MLH1, MSH2, MSH6, PMS2, and EPCAM (9, 10). Most of the pathogenic variants of MMR genes include small insertions/deletions or large genetic rearrangements, missense, and nonsense variants (11–13). The majority of these gene defects were found in MLH1 and MSH2 (60 to 80%) genes. Other detrimental variants in MSH6 or PMS2 and, rarely, to EpCAM genes have been identified associated with Lynch phenotype. Nevertheless, recent population studies have found a higher frequency for germline pathogenic variants in MSH6 and PMS2 genes (14–16). It was demonstrated that these genes encompass lower penetrance pathogenic variants, and MSH6 deleterious variant carriers represent late-onset cancer compared to MLH1 or MSH2 carriers (13). Moreover, an increased risk of extracolonic tumors, particularly endometrial cancer, has been shown in MSH2 pathogenic variant carriers (13). Endometrial and ovarian cancers are the most prevalent Lynch-associated tumors. Other associated cancers include pancreatic, biliary tract, ureter, renal pelvis, gastric, small intestine, brain, sebaceous adenomas, sebaceous carcinomas, and keratoacanthomas (9).

LS is also known as hereditary non-polyposis colorectal cancer (HNPCC), with an approximate prevalence of one in 600 to one in 3,000 individuals (17, 18). HNPCC is a heterogeneous group of disorders with overlapping features that are clinically evaluated using Amsterdam (I or II) or Bethesda guidelines (19–22). Clinical guidelines are based on family and personal history of cancer, and their use as diagnostic tools for LS is restricted due to their low sensitivity. Patients with HNPPCC are at increased risk for metachronous or synchronous colon and extracolonic Lynch-related cancers. Since many patients with Lynch syndrome (UP TO 50%) do not even meet the revised Bethesda guidelines, an alternative approach referred to as “universal tumor screening” is recommended. Both MMR immunohistochemistry (IHC) staining and MSI analyses (either by themselves or in combination) are used to test MMR system proteins in all newly diagnosed CRC patients (9, 10). MSI is not exclusive to the Lynch-related CRCs. It may be observed in other CRC tumors. Somatic MLH1 gene inactivation due to promoter hypermethylation characterizes sporadic MMR deficient (dMMR) colorectal tumors, which typically arise in patients with late-onset cancer (12, 13). Unexplained dMMR CRC, termed “Lynch-like syndrome”, is another MSI colorectal tumor for which neither the deleterious germline variant of MMR genes nor the hypermethylation of the MLH1 gene promoter can be identified (12, 15, 23). Somatic bi-allelic mutations in the MMR genes may explain a large number of these Lynch-like dMMR tumors (nearly 70%). However, the etiology of a large number of these unclassified MSI/dMMR tumors is unclear (12, 23, 24). Moreover, dMMR CRCs encompass around 60% of HNPCC cases. About 40% of HNPCC patients do not represent MMR defects, and they are accordingly introduced as familial colorectal cancer type X (FCCTX) with undefined etiology. FCCTX is a group of heterogeneous diseases characterized by microsatellite stable (MSS) and late-onset clinically different from LS (21, 25, 26). Despite the recent advances in gene identification technologies, the genetic etiology of many hereditary cancers remains undefined, especially FCCTX groups (27, 28).

By the poor clinical diagnostic approaches, it is difficult to differentiate Lynch CRC tumors from non-hereditary ones due to the high frequency of MSI occurrence in sporadic tumors. This situation becomes more complicated when there is a positive family history of developing CRC or different related cancer types (19). In the present study, a CRC-affected individual from a fulfilled Amsterdam II criteria pedigree, showing Lynch-associated MSI and IHC phenotypes, underwent whole-exome sequencing (WES). The analysis of WES data revealed no pathogenic variants in any of the MMR genes. Further evaluation unveiled a frameshift variant in WRN (RECQL2; HGNC ID: 12791) gene as a potential candidate gene deserving subsequent considerations.

WRN encodes a DNA helicase enzyme from the highly conserved RECQ gene family and has a pivotal role in DNA repair and maintenance of genomic stability (29, 30). The WRN has both helicase and exonuclease activity and has been considered a genomic caretaker (31). Homozygous truncating variants in the WRN gene cause Werner syndrome (WS; OMIM 277700) manifests, which are characterized by early onset age-associated diseases such as cancer (32, 33). In addition to the elevated risk of developing cancer in WS patients, several studies have indicated a tumor suppressor-like function for WRN, while making it a prominent cancer-predisposing gene (34, 35). More importantly, various evaluations have suggested a crucial role for WRN associated with different types of familial cancers, specifically breast and ovarian cancers (36–38). However, the association of germline WRN gene variants with familial CRC has not been established well. Herein, we aimed to introduce a truncating variant in WRN (NM_000553:exon9:p.R389Efs*3) as a pathogenic candidate based on ACMG standard guideline (39) in a patient affected by early onset familial CRC.

While many cancers have a positive family history, pathogenic variants in the well-known cancer-predisposition genes are responsible for up to 10% of hereditary cancers. The number of genes involved in cancer susceptibility is growing and further clinical and molecular evidence is needed to explain the physiopathology of these variants in cancer (47).

In the present study, we investigated the genetic etiology of the familial CRC in a pedigree meeting the Amsterdam criteria (48, 49). Since the clinical and pathological data and molecular analysis strongly implicated the LS (41), we originally focused on the evaluation of MMR system gene variants. Furthermore, we performed Sanger sequencing to assess the presence of any candidate pathogenic variant in the MSH2 and MSH6 gene promoters (43, 45). According to the InSIGHT database and the ACMG guideline, no pathogenic variant was identified in the exonic and regulatory elements of MMR genes. Moreover, MLPA testing was done. The data ruled out the presence of deleterious duplication or deletion in MLH1 or MSH2 genes (50). Accordingly, reanalysis of exome data through GeneCard and COSMIC dataset resulted in a list of relevant gene variants ( Table 1 ). The subsequent assessment suggested that a WRN frameshift pathogenic variant (p.R389Efs*3) could be related to the etiology. Interestingly, the proband’s maternal aunt, II:8, suffering from breast cancer, was found to carry this variant.

Although WRN has been introduced as a candidate gene for hereditary breast cancer (37), the inheritance of the variant from the maternal side has sparked a debate on its role in developing colorectal cancer. The observation of dMMR phenotype might suggest the possibility of developing sporadic CRC due to bi-allelic somatic inactivation of MSH2 and MSH6 genes (12, 24). However, the positive history of cancer in this family and the patient’s young age weaken this assumption in the proband. Zimmer et al. showed that 56% of CRC tumors with bi-allelic mutant WRN gene presented MSI-H/dMMR status (51). The wide spectrum of reported cancers related to different tumor suppressor genes (BRCA1, BRCA2, MSH2, MLH1, etc.) would support the hypothesis that WRN, which has been previously confirmed as a candidate gene for hereditary breast cancer, may also lead to CRC (52, 53).

However, there remains a slight chance for the inheritance of an MMR gene pathogenic variant from the paternal side possibly not detected through the applied methods in the present study. According to several studies, MSI has a multi-pathway mechanism, and non-mutational events and implications of novel genes may explain MSI/dMMR tumors with no detectable germline mutation (32, 54, 55). For example, it was demonstrated that BCL2 gene overexpression, commonly expressed in various tumor types, can downregulate the MSH2 gene and subsequently reduce MMR protein cellular activity via the hypophosphorylation of pRb protein (56). Likewise, it was revealed that WRN gene silencing is linked to the MSI in CRC tumors and in vitro MSI models (54, 57). In this regard, WRN plays an essential role in the survival of MSI/dMMR cancer cell lines (not in MMR-proficient and MSS cells). It has been proposed that WRN is involved in an EXO1 independent pathway for mismatch repair in MSI-H cells (58, 59). Besides, MSI-H/dMMR high frequency in the WRN mutant CRC tumors would support this hypothesis that WRN may be involved in the MSI events as an alternative pathway in undefined dMMR CRCs (51). Our finding in the present study will put forth WRN as a novel gene possibly leading to CRC associated with MSI, although its exact mechanism remains unknown.

The identified variant was interpreted as pathogenic based on the ACMG guideline. Since this premature stop codon variant disrupted the helicase domain, the protein function is lost (PVS1) (39, 60). The frequency of this variant was notably low in genomeAD-Exome Asian and ExAC Asian databases and even absent in genomeAD-genome East Asian population data (PM2). This variant was represented in ClinVar (Variation ID: 238117) associated with WS (PS3). The functional assessments indicated that the damaging effects on WRN gene products were attributed to genomic instability in colorectal tumor cells (35). Furthermore, aberrant WRN function due to hemizygous gene loss was demonstrated in advanced clinical CRC (61). In addition, the genetic instability revealed in cell lines from WS patients indicates haploinsufficiency for the WRN gene (32).

The defective WRN, a member of the RecQ helicase family, implicates WS (29). RecQ helicases are known as genome caretakers and play a critical role in genomic stability (62). The involvement of WRN, BLM, and RecQL4 family members has been determined in the pathogenesis of well-defined genetic disorders with an elevated risk of cancer (63). The WRN gene is located at 8p12 and contains 35 coding exons. The protein comprises ATPase, RQC, and HRDC domains, as well as an N-terminal exonuclease domain. It was shown that WRN participates in several DNA metabolic pathways, including replication, recombination, and repair to preserve the genome (30, 31, 64). To date, 140 pathogenic and 31 likely pathogenic variants have been identified in the WRN gene based on the ClinVar database. These variants include deletion, duplication, indel, insertion, and single nucleotide substitution leading to the destabilizing or protein failure to localization to the nucleus. The recessive truncating variants are described in association with WS well, suggesting that complete loss of WRN is necessary to develop the disease (32).

It was determined that truncating variants, which destroy the helicase activity of WRN, participate in WS pathogenesis (32, 60). The complete loss of WRN in WS patients born with a germline homozygous deleterious variant will lead to premature senescence and neoplastic alteration (65). WRN inactivation can induce DNA double-strand breaks and subsequently activate DNA damage responses such as apoptosis and cell cycle arrest (57). WS patients are susceptible to a variety of cancer types, including colorectal, ovarian, pancreatic, skin, and breast cancers, as well as soft tissue sarcomas, osteosarcomas, and leukemia (65, 66). A common pathogenic variant was shown to play a pivotal role in the genesis of thyroid carcinoma in Japanese WS patients. Furthermore, the association of the follicular and papillary carcinomas with the WRN c.3139-1G>C (exon 26 skip) and c.1105C>T (p.R369*) variants was found in WS patients, respectively (67, 68). Considering the significant frequency of different neoplasms in WS patients, WS was considered a heritable cancer predisposition syndrome. However, limited studies have investigated the role of WRN in developing cancer in non-WS individuals. Experiments revealed a degree of genetic instability for carriers of the WRN heterozygous pathogenic variants. These studies demonstrated that WRN inactivation promotes genetic instability in different human somatic cell lineages (32, 54, 68). However, it should be noted that WRN has a tumor suppressor function, and the loss of its second wild-type allele is necessary for carcinogenesis events to fulfill Knudson’s two-hit hypothesis of tumor suppressor genes (11).

Notably, epigenetic inactivation of the WRN gene was introduced as a common event in sporadic colorectal tumorigenesis as described for other DNA repair tumor suppressor genes, hMLH1 and BRCA1, involved in hereditary cancers (34). Furthermore, the aberrant function of the WRN gene in MSI-H CRC tumors, as a result of frameshift variants, suggested the probability of WRN contribution at MSI-H tumorigenesis as a putative tumor suppressor (35). Additionally, WRN’s contribution to breast cancer susceptibility has been demonstrated by Ding et al. They reported that a WRN specific variant (rs9649886), situated in the C-terminal of WRN, significantly affected breast cancer risk (36). Likewise, Zins et al. examined the significance of a WRN non-synonymous variant (rs1346044) with an elevated risk of breast cancer. They indicated that the risk of breast cancer increased in homozygous individuals due to the reduced helicase activity (38). Even though the potential role of WRN in promoting breast cancer was undetermined, more recently, a novel germline frameshift variant (p.N1370Tfs*23) has been identified in a Chinese family. The truncated variant was confirmed in three affected individuals and an unaffected first-degree relative. The identified variant was interpreted as pathogenic according to the ACMG guidelines, thereby introducing WRN as a potential candidate gene for hereditary breast cancer (37, 39).

In conclusion, hereditary cancer syndromes have provided a remarkable venue in cancer investigation, particularly in diagnostic and therapeutic strategies. Furthermore, the identification of at-risk individuals through germline mutation testing is a key to success in the prevention, early detection, and management of patients (21, 26). However, WRN has not been addressed as a hereditary CRC predisposing gene so far. We noticed a WRN pathogenic variant as a disease-causing variant in a familial CRC patient. Our data could be further authenticated by following up the family, performing additional functional studies, and precisely detecting the loss of heterozygosity feature in colorectal tumor cells. Nevertheless, the potential role of WRN in CRCs remains elucidated, and further studies are needed to reveal its role in hereditary cancers, HNPCC subtypes in particular.

The original contributions presented in the study are included in the article/ Supplementary Material ; further inquiries can be directed to the corresponding author/s.

The study involving human participants was reviewed and approved by the Ethics Committee of Shahid Chamran University of Ahvaz (Ref. EE/97.24.3.70385/scu.ac.ir). The patients/participants provided their written informed consent to participate in this study.

MS and MT conceived and designed the study, obtained funding, developed methodology, reviewed, and revised the manuscript. MZ and ME managed patients and provided facilities. MN drafted the manuscript. HG revised the manuscript. MN, ZA, and PM performed molecular techniques and interpretation of data. All authors contributed to the article and approved the submitted version.

Shahid Chamran University of Ahvaz (grant number 97/3/02/26247) and Isfahan University of Medical Science (grant number 195216) financially supported this work.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.